Seasonal influenza outbreaks across the globe cause an estimated 250,000 to 500,000 deaths annually. Current influenza vaccines need to be updated every few years because of antigenic drift (Pica el al., Annu Rev Med., 2013, 64,189-202). Despite intensive monitoring, strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past (Carrat et al., Vaccine, 2007, 25(39-40), 6852-6862). Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift facilitating a potential pandemic outbreak. These concerns have expedited efforts towards developing a ‘universal’ flu vaccine.
Neutralizing antibodies (nAbs) against hemagglutinin (HA) are the primary correlate for protection in humans and hence HA is an attractive target for vaccine development (Gerhard, Curr Top Microbiol Immunol., 2001, 260,171-190). The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the disulfide linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1. subunit while the stem domain predominantly comprises of the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH induced conformational rearrangement in the host-cell endosomes to adopt the virus-host membrane fusion competent state (Carr et al., Cell, 1993, 73(4), 823-832; Skehel et al., Annu Rev Biochem., 2000, 69, 531-569).
The antigenic sites on the globular head of HA are subjected to heightened immune pressure resulting in escape variants; thereby limiting the breadth of head-directed nAbs (Knossow et al., Immunology, 2006, 119(1), 1-7). However, extensive efforts have resulted in the isolation of monoclonal antibodies which bind within the globular head and inhibit receptor attachment, which neutralize drifted variants of an HA subtype or heterosubtypic HA subtype (Ekiert D C, et al.,Nature, 2012, 489(7417), 526-532; Hong M, et al. J Virol., 2013, 87(22), 12471-12480; Krause J C, et al., J Virol., 2011, 85(20), 10905-10908; Lee P S, et al., Proc Natl Acad Sci USA, 2012, 109(42), 17040-17045; Ohshima N, et al., J Virol., 2011, 85(21), 11048-11057; Schmidt A G, et al., Proc Natl Acad Sci USA, 2013, 110(1), 264-269; Tsibane T, et al., PLoS Pathog, 2012, 8(12), e1003067; Whittle J R, et al., Proc Natl Acad Sci USA, 2011, 108(34), 14216-14221; Wrammert J, et al., J Exp Med., 2011, 208(1), 181-193; Xu R, et al., Nat Struct Mol Biol., 2013, 20(3), 363-370).
The HA stem is targeted by several neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes (Julien J P et al., Immunol Rev., 2012, 250(1), 180-198). The epitopes of these bnAbs in the HA stem are more conserved across different influenza HA subtypes compared to the antigenic sites in the HA globular head (Ellebedy A H et al., Front Immunol., 2012, 3, 53).
A ‘headless’ stem domain immunogen offers an attractive solution. However, early attempts at expressing the HA2-subunit independently in a native, pre-fusion conformation have been unsuccessful. In the absence of the head domain, the HA2-subunit expressed in E. coli spontaneously adopts a low-pH conformation (Chen J, et al., Proc Natl Acad Sci USA, 1995, 92(26), 12205-12209) in which the functional epitopes of stem-directed bnAbs are disrupted. More recently, the entire HA stem region has been expressed in a pre-fusion, native-like conformation in both prokaryotic and eukaryotic systems adopting multiple strategies (Bommakanti G, et al., Proc Natl Acad Sci USA, 2010, 107(31), 13701-1370; Bommakanti G, et al., J Virol, 2012, 86(24), 13434-13444; Lu Y et al., Proc Natl Acad Sci USA, 2014, 111(1), 125-130; Steel J, et al., M Bio., 2010, 1(1)).
U.S. Pat. No. 6,720,409 provides an anti-human influenza virus antibody which recognizes the stem regions of haemagglutinin molecules of the H1N1 and H2N2 subtypes and has a neutralization activity but does not recognize the stem region of the H3N2 subtype and has no neutralization activity.
WO2013011347 describes antibodies, and antigen binding fragments thereof, that specifically bind to an epitope in the stem region of an influenza A hemagglutinin trimer and neutralize a group I subtype and a group 2 subtype of influenza A virus.
U.S. Pat. No. 5,589,174describes an anti-human influenza virus antibody is provided having the following characteristics: (a) specifically binds to the stem region of hemagglutinin of human influenza A virus subtype H3N2; (b) does not specifically bind to the stem region of hemagglutinin of human influenza A virus subtypes H1N1 and H2N2; and (c) does not specifically bind to the stem region of hemagglutinin of human influenza B virus.
WO201377444 describes vaccine compositions and methods of producing and using the same, which compositions comprise a modified HA stem domain in trimeric configuration.